The present invention relates generally to methods and systems for examining biological materials and more particularly to methods and systems for examining biological materials using Raman spectroscopy.
A variety of Raman spectroscopic techniques have been reported in the art as being either useful or potentially useful in medical applications. For example, in U.S. Pat. No. 5,261,410, inventors Alfano et al., which issued Nov. 16, 1993, and which is incorporated herein by reference, there is disclosed a method for determining if a tissue is a malignant tumor tissue, a benign tumor tissue, or a normal or benign tissue using Raman spectroscopy. The method is based on the discovery that, when irradiated with a beam of infrared, monochromatic light, malignant tumor tissue, benign tumor tissue, and normal or benign tissue produce distinguishable Raman spectra. For human breast tissue, some salient differences in the respective Raman spectra are the presence of four Raman bands at a Raman shift of about 1078, 1300, 1445 and 1651 cmxe2x88x921 for normal or benign tissue, the presence of three Raman bands at a Raman shift of about 1240, 1445 and 1659 cmxe2x88x921 for benign tumor tissue, and the presence of two Raman bands at a Raman shift of about 1445 and 1651 cmxe2x88x921 for malignant tumor tissue. In addition, it was discovered that, for human breast tissue, the ratio of intensities of the Raman bands at a Raman shift of about 1445 and 1650 cmxe2x88x921 is about 1.25 for normal or benign tissue, about 0.93 for benign tumor tissue, and about 0.87 for malignant tumor tissue.
As another example, in U.S. Pat. No. 5,293,872, inventors Alfano et al., which issued Mar. 15, 1994, and which is incorporated herein by reference, there is disclosed a method for distinguishing between, on one hand, calcified atherosclerotic tissue and, on the other hand, fibrous atherosclerotic tissue or normal cardiovascular tissue using Raman spectroscopy. The method is based on the discovery that, when irradiated with a beam of monochromatic infrared light, calcified atherosclerotic human aortic tissue produces a Fourier Transform Raman spectrum which is distinguishable from analogous spectra obtained from fibrous atherosclerotic human aortic tissue mad normal human aortic tissue. Some salient differences in the respective Raman spectra are the presence of five Raman bands at Raman shifts of 957, 1071, 1262-1300, 1445, and 1659 cmxe2x88x921 (xc2x14 cmxe2x88x921 for all shifts) for the calcified tissue as compared to three Raman bands at Raman shifts of 1247-1270, 1453 and 1659 cmxe2x88x921 (xc2x14 cmxe2x88x921 for all shifts) for the fibrous tissue and three Raman bands at Raman shifts of 1247-1270, 1449 and 1651 cmxe2x88x921 (xc2x14 cmxe2x88x921 for all shifts) for the normal tissue. In addition, it was discovered that the ratios of intensities for the Raman bands at 1659 and 1453 cmxe2x88x921 and at 1254 and 1453 cmxe2x88x921 were 0.69 and 0.53, respectively, for the calcified tissue, 1.02 and 0.85, respectively, for the fibrous tissue and 1.2 and 0.83, respectively, for the normal tissue.
As yet another example, in U.S. Pat. No. 5,243,983, inventors Tarr et al., which issued Sep. 14, 1993, and which is incorporated herein by reference, there is disclosed a non-invasive blood glucose measurement system and method using stimulated Raman spectroscopy. The system and method make use of two monochromatic laser beams, a pump laser beam and a probe laser beam. The output power of the pump laser beam is amplitude modulated, combined with the probe laser beam and directed into the ocular aqueous humor of a living being. The introduction of the laser beams into the ocular aqueous humor induces scattered Raman radiation, which causes a portion of the energy at the pump frequency to shift over to the probe frequency. The pump and probe laser beams are then detected as they exit the ocular aqueous humor. The probe laser beam is filtered, converted into an electrical signal and amplified. It is then compared to the modulation signal to generate an electrical signal representative of the concentration of D-glucose in the ocular aqueous humor.
Some of the problems with Raman spectroscopic techniques of the type described above (e.g., spontaneous Raman spectroscopy, coherent anti-stokes Raman spectroscopy and pulse-pumped stimulated Raman spectroscopy) are that such techniques typically require a long exposure time (i e., several minutes) and high power laser pump fluency that exceeds the safety limitations for laser illumination in human body applications. As can readily be appreciated, requirements such as these substantially limit the practical applicability of these techniques in the fields of in vivo and in vitro medical diagnostics.
Other documents of interest include Owyoung, xe2x80x9cCoherent Raman Gain Spectroscopy Using CW Laser Sources,xe2x80x9d IEEE Journal of Quantum Electronics, QE-14(3):192-202 (1978); Liu et al., xe2x80x9cRaman, fluorescence, and time-resolved light scattering as optical diagnostic techniques to separate diseased and normal biomedical media, J. Photochem. Photobiol. B: Biol., 16:187-209 (1992); Lowenstein et al., xe2x80x9cNitric Oxide: A Physiologic Messenger,xe2x80x9d Ann Intern Med., 120:227-237 (1994); and Berger et al., xe2x80x9cFeasibility of measuring blood glucose concentration by near-infrared Raman spectroscopy,xe2x80x9d Spectrochimica Acta Part A, 53:287-292 (1997), all of which are incorporated herein by reference.
It is an object of the present invention to provide a novel method and system for examining biological materials using Raman spectroscopy.
It is another object of the present invention to provide a method and system as described above that overcome at least some of the problems associated with existing Raman spectroscopic methods and systems for examining biological materials.
It is still another object of the present invention to provide a method and system as described above that may be used in in vivo and/or in vitro medical diagnostics.
In accordance with the above objects, as well as with those objects to become apparent from the description to follow, there is hereinafter disclosed a novel method and system for examining biological materials, said method and system employing a low-power continuous wave (cw) pump laser beam and a low-power cw Stokes (or anti-Stokes) probe laser beam. The pump beam and the probe beam simultaneously illuminate the biological material and traverse the biological material in collinearity. The pump beam, whose frequency is varied, is used to induce Raman emission from the biological material. The intensity of the probe beam, whose frequency is kept constant, is monitored as it leaves the biological material. When the difference between the pump (xcexa9) and probe (xcfx89) excitation frequencies is equal to a Raman vibrational mode frequency (xcexd) of the biological material, the weak probe signal becomes amplified by one or more orders of magnitude (typically up to about 104-106) due to the Raman emission from the pump beam. In this manner, by monitoring the intensity of the probe beam emitted from the biological material as the pump beam is varied in frequency, one can obtain an excitation Raman spectrum for the biological material tested.
The present invention may be applied to in the in vivo and/or in vitro diagnosis of diabetes, heart disease, hepatitis, cancers and other diseases by measuring the characteristic excitation Raman lines of blood glucose, cholesterol, serum glutamic oxalacetic transaminase (SGOT)/serum glutamic pyruvic transaminase (SGPT), tissues and other corresponding Raman-active body constituents, respectively. For example, it may also be used to diagnose diseases associated with the concentration of Raman-active constituents in urine, lymph and saliva. It may be used to identify cancer in the breast, cervix, uterus, ovaries and the like by measuring the fingerprint excitation Raman spectra of these tissues. It may also be used to reveal the growing of tumors or cancers by measuring the levels of nitric oxide in tissue.
Some of the advantages of the present invention are that the subject method can be performed in a short detecting time (i.e., on the order of several seconds as opposed to several minutes) and that the pump and probe beams can be operated at a power of up to four orders of magnitude less than that typically used in pulsed coherent Raman spectroscopic techniques.
Additional objects, features, aspects and advantages of the present invention will be set forth, in part, in the description which follows and, in part, will be obvious from the description or may be learned by practice of the invention. In the description, reference is made to the accompanying drawings which form a part thereof and in which is shown by way of illustration specific embodiments for practicing the invention. These embodiments will be described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that structural changes may be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is best defined by the appended claims.